rsos.royalsocietypublishing.org Research Cite this article: Reddon AR, O’Connor CM, Marsh-Rollo SE, Balshine S, Gozdowska M, Kulczykowska E. 2015 Brain nonapeptide levels are related to social status and affiliative behaviour in a cooperatively breeding cichlid fish. R. Soc. open sci. 2: 140072. http://dx.doi.org/10.1098/rsos.140072 Received: 1 June 2014 Accepted: 7 January 2015 Subject Category: Biology (whole organism) Subject Areas: behaviour Keywords: isotocin, arginine vasotocin, oxytocin, vasopressin, dominance, Neolamprologus pulcher Author for correspondence: Adam R. Reddon e-mail: [email protected]† Present address: Department of Biology, McGill University, 845 Sherbrooke Street West, Montreal, Quebec, Canada H3A 0G4. Electronic supplementary material is available at http://dx.doi.org/10.1098/rsos.140072 or via http://rsos.royalsocietypublishing.org. Brain nonapeptide levels are related to social status and affiliative behaviour in a cooperatively breeding cichlid fish Adam R. Reddon 1,† , Constance M. O’Connor 1 , Susan E. Marsh-Rollo 1 , Sigal Balshine 1 , Magdalena Gozdowska 2 and Ewa Kulczykowska 2 1 Aquatic Behavioural Ecology Laboratory, Department of Psychology, Neuroscience, and Behaviour, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1 2 Genetics and Marine Biotechnology, Institute of Oceanology of Polish Academy of Sciences, Powstanców Warszawy 55 Street, 81-712 Sopot, Poland 1. Summary The mammalian nonapeptide hormones, vasopressin and oxytocin, are known to be potent regulators of social behaviour. Teleost fishes possess vasopressin and oxytocin homologues known as arginine vasotocin (AVT) and isotocin (IT), respectively. The role of these homologous nonapeptides in mediating social behaviour in fishes has received far less attention. The extraordinarily large number of teleost fish species and the impressive diversity of their social systems provide us with a rich test bed for investigating the role of nonapeptides in regulating social behaviour. Existing studies, mostly focused on AVT, have revealed relationships between the nonapeptides, and both social behaviour and dominance status in fishes. To date, much of the work on endogenous nonapeptides in fish brains has measured genomic or neuroanatomical proxies of nonapeptide production rather than the levels of these molecules in the brain. In this study, we measure biologically available AVT and IT levels in the brains of Neolamprologus pulcher, a cooperatively breeding cichlid fish, using high performance liquid chromatography with fluorescence detection. We found that brain AVT levels were higher in the subordinate than in dominant animals, and levels of IT correlated negatively with the expression of affiliative behaviour. We contrast these results with previous studies, and we discuss the role the nonapeptide hormones may play in the regulation of social behaviour in this highly social animal. 2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.
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ResearchCite this article: Reddon AR, O’Connor CM,Marsh-Rollo SE, Balshine S, Gozdowska M,Kulczykowska E. 2015 Brain nonapeptide levelsare related to social status and affiliativebehaviour in a cooperatively breeding cichlidfish. R. Soc. open sci. 2: 140072.http://dx.doi.org/10.1098/rsos.140072
†Present address: Department of Biology,McGill University, 845 Sherbrooke Street West,Montreal, Quebec, Canada H3A 0G4.
Electronic supplementary material is availableat http://dx.doi.org/10.1098/rsos.140072 or viahttp://rsos.royalsocietypublishing.org.
Brain nonapeptide levelsare related to social statusand affiliative behaviourin a cooperatively breedingcichlid fishAdam R. Reddon1,†, Constance M. O’Connor1,
Susan E. Marsh-Rollo1, Sigal Balshine1,
Magdalena Gozdowska2 and Ewa Kulczykowska2
1Aquatic Behavioural Ecology Laboratory, Department of Psychology, Neuroscience,and Behaviour, McMaster University, 1280 Main Street West, Hamilton, Ontario,Canada L8S 4K12Genetics and Marine Biotechnology, Institute of Oceanology of Polish Academy ofSciences, PowstancówWarszawy 55 Street, 81-712 Sopot, Poland
1. SummaryThe mammalian nonapeptide hormones, vasopressin andoxytocin, are known to be potent regulators of social behaviour.Teleost fishes possess vasopressin and oxytocin homologuesknown as arginine vasotocin (AVT) and isotocin (IT), respectively.The role of these homologous nonapeptides in mediatingsocial behaviour in fishes has received far less attention. Theextraordinarily large number of teleost fish species and theimpressive diversity of their social systems provide us with a richtest bed for investigating the role of nonapeptides in regulatingsocial behaviour. Existing studies, mostly focused on AVT, haverevealed relationships between the nonapeptides, and both socialbehaviour and dominance status in fishes. To date, much of thework on endogenous nonapeptides in fish brains has measuredgenomic or neuroanatomical proxies of nonapeptide productionrather than the levels of these molecules in the brain. In thisstudy, we measure biologically available AVT and IT levels inthe brains of Neolamprologus pulcher, a cooperatively breedingcichlid fish, using high performance liquid chromatographywith fluorescence detection. We found that brain AVT levelswere higher in the subordinate than in dominant animals, andlevels of IT correlated negatively with the expression of affiliativebehaviour. We contrast these results with previous studies, andwe discuss the role the nonapeptide hormones may play in theregulation of social behaviour in this highly social animal.
2015 The Authors. Published by the Royal Society under the terms of the Creative CommonsAttribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricteduse, provided the original author and source are credited.
................................................2. IntroductionUnderstanding the mechanistic underpinnings of social behaviour represents a major research goalin behavioural biology [1–3]. The nonapeptide hormones, oxytocin and vasopressin, have attracteda great deal of research attention, and there is strong evidence that these neurohormones are keyproximate regulators of mammalian social behaviour [4–9]. These neuropeptides are evolutionarilyancient molecules that trace back to the original nonapeptide, arginine vasotocin (AVT), which is foundin extant non-mammalian vertebrates [10]. Oxytocin, which originally arose from a duplication of theAVT gene early in the vertebrate lineage, also has non-mammalian homologues in all extant vertebrates,including isotocin (IT) in teleost fishes and mesotocin in birds, amphibians and non-avian reptiles [10,11].Much less research attention has been dedicated to the role that these homologous molecules play in theregulation of social behaviour in non-mammalian vertebrates, though in recent years, this has begunto change, notably with work that has been done linking AVT and mesotocin to social behaviour inbirds [12–15].
Teleost fishes represent by far the most diverse group of living vertebrates [16] and exhibit asimilarly diverse range of social systems [17]. Teleost fishes, therefore, present an excellent opportunityto investigate the role of the nonapeptide hormones in regulating social behaviours [18]. Furthermore,extant teleosts may provide a window into the ancestral role of nonapeptide circuits in regulating socialbehaviour, and offer insights into the degree to which nonapeptide hormone function has been conservedthrough vertebrate evolution [10,19]. Despite the remarkable diversity of the teleost fishes, relatively littlework has been done to investigate how these nonapeptide hormones influence the social behaviour offishes [18,19].
The relatively small number of experiments that have manipulated AVT and IT levels in fisheshave provided some clues as to the function of these neuropeptides in regulating social behaviour.For example, nonapeptides appear to regulate social approach behaviour in fishes. Goldfish (Carassiusauratus) that received an intracerebroventricular infusion of AVT spent less time associating with aconspecific, whereas fish that received an administration of an AVT antagonist spent more time neara conspecific [20]. An intracerebroventricular infusion of IT also increased social approach in the samespecies [20], suggesting that both AVT and IT play a role in the regulation of social approach in goldfish.Consistent with that conclusion, both AVT and IT affect social approach when given peripherally tozebrafish (Danio rerio), with the direction of those effects being dose-dependent [21]. The administrationof exogenous AVT increases aggression in bluehead wrasse (Thalassoma bifasciatum [22]), brown ghostknifefish (Apteronotus leptorhynchus [23]) and beaugregory damselfish (Stegastes leucostictus [24]), butdecreases aggression in Amargosa River pupfish (Cyprinodon nevadensis amargosae [25]) and zebrafish(Danio rerio [26]). Administrations of IT, by contrast, seem to have little effect on the absolute level ofaggressive behaviour in fishes [24,27]. Nonapeptide administrations also have effects on pair bondingand parental care behaviours in the monogamous convict cichlid (Amatitlania nigrofasciata). A non-specific AVT/IT antagonist delivered peripherally delayed, but did not prevent, pair bonding in convicts[28], whereas a selective IT antagonist interfered with parental care [29].
Most of the previous studies in fishes have used indirect measures of nonapeptide concentrationsbased on neuroanatomy or mRNA expression to examine the relationship between endogenousnonapeptide levels in the brain and social behaviour. These studies have used the techniques suchas immunohistochemistry, in situ hybridization or qPCR. These studies have provided some insightsinto the relationship between endogenous nonapeptides in the brain and social behaviour in fishes,and have shown that individual neuronal phenotypes are predictive of differences in behaviour anddominance status. For example, in the plainfin midshipman, Porichthys notatus, dominant territorialmales have larger AVT-immunoreactive cells in their preoptic area compared with females or sneakermales [30]. In the beaugregory damselfish, AVT fibre density in the parvocellular region of thepreoptic area is inversely related to aggression [31]. In the polygynous African cichlid, Astatotilapiaburtoni, dominant males show greater AVT mRNA expression in the gigantocellular region of theirpreoptic area, whereas subordinate males have greater expression in the parvocellular region of thepreoptic area [32]. Interestingly, AVT expression in dominant males correlates positively with aggressivebehaviour, whereas AVT expression in the subordinate males correlates positively with submissivebehaviour [32].
Recently, high performance liquid chromatography with florescence detection (HPLC-FL) hasbeen used to measure the levels of nonapeptide hormones directly within fish brains [33]. Suchmeasurement of biologically available molecules may be more straightforwardly related to the currentneuromodulatory actions of nonapeptides in the animal’s brain compared with indirect proxies of
................................................nonapeptide production. Using this technique, fishes have been shown to differ in the amount ofAVT and IT in their brains depending on dominance and reproductive status. Subordinate Oreochromismossambicus had higher levels of AVT in their pituitary and higher levels of IT in their hindbrain thandid dominant fish [34]. Conversely, both AVT and IT levels were higher in whole brain preparationsof dominant three-spined stickleback males, Gasterosteus aculeatus, relative to socially subordinateanimals [35]. Moreover, female three-spined sticklebacks housed alone have higher AVT and lower ITconcentrations in their brains relative to females housed in groups, whereas males housed alone haveincreased AVT levels and show no differences in IT concentrations relative to males housed in groups[36]. Biologically available nonapeptide levels in the brain also differ depending on social housingdensity in the round goby (Neogobius melanostomus), where non-aggressive males held at high densitiesshow higher whole brain AVT and lower whole brain IT levels than territorial males housed at lowerdensity [37].
In order to further explore the relationship between nonapeptide levels in fish brains and socialbehaviour, we made use of an emerging model system in the integrative biology of social behaviour,the cooperatively breeding cichlid fish, Neolamprologus pulcher. Neolamprologus pulcher are a substratespawning cichlid fish endemic to the rocky littoral zone in Lake Tanganyika, East Africa [38].Neolamprologus pulcher exhibit a complex social system characterized by frequent social interactions anda suite of specialized social behaviours and signals [39–43]. Neolamprologus pulcher live and breed withinsocial groups comprising a single dominant breeding pair and 1–20 non-breeding adult subordinatesof both sexes [42,44]. Subordinate N. pulcher assist the reproductive efforts of the dominant pair byclearing the territory of sand and debris, defending against predators, conspecific and heterospecificspace competitors, and participating in care of the young (for review, see [43]). The exceptional socialnature of this fish combined with its tractability for controlled laboratory experiments makes N. pulcheran attractive study system for unravelling the role the nonapeptide hormones play in regulating socialbehaviour [27]. In particular, N. pulcher are small-bodied (maximum size is approx. 80 mm standardlength), fast-growing and adapt well to life in aquaria [40,45] where they form naturalistic social groupsand perform their full suite of social behaviours [46].
Previous studies examining the functions of the nonapeptides in N. pulcher suggest that thesehormones play an important role in regulating social behaviour in this species. In a microarray study,dominant N. pulcher showed higher expression of the AVT gene than did subordinates, suggesting thatAVT plays a role in determining social status in this species [47]. Reddon et al. [27] found that N. pulchertreated with an intraperitoneal injection of IT showed more submissive displays within their socialgroups. Submissive displays are an important social signal in this species, which may serve to appeasedominant group members [48] and facilitate conflict resolution while allowing both parties to remainin the same spatial location [49], suggesting a possible role for IT in the regulation of the dominancehierarchy. Reddon et al. [50] found that exogenous IT reduced the tendency for N. pulcher to approachand affiliate with conspecifics, in contrast to previous work in goldfish [20].
In this study, we investigated the relationship between nonapeptide hormones, dominance status andsocial behaviour in N. pulcher. Following behavioural observations, we measured the concentrations ofbiologically available nonapeptides, AVT and IT, in the brains of dominant and subordinate N. pulcher ofboth sexes using HPLC-FL. The results of this study complement previous work analysing nonapeptidegene expression and manipulating nonapeptides in N. pulcher [27,47,50] and together offer a morecomplete picture of the role of the nonapeptide hormones in regulating social behaviour in this highlysocial fish.
3. Material and methods3.1. Study animalsAll of the fish that we used in this study were adults from a breeding colony of N. pulcher held atMcMaster University, Hamilton, Ontario, Canada. The fish were descendants of breeding pairs caught inLake Tanganyika, Zambia in 2002 and 2008 and had been housed in the laboratory for several generationsprior to this study. The fish lived in social groups consisting of a male and female dominant breedingpair with two subordinate helpers. These social groups had been together for at least one month prior tothe onset of this study, and all groups had successfully reproduced at least once before being includedin this study. Therefore, these represent stable social groups. Each social group was housed in a 189 lfreshwater tank outfitted with a heater, thermometer, two foam filters, approximately 2 cm of coral sand
................................................substrate, and two inverted flowerpot halves for use as shelters and nest sites. The light : dark cycle waskept constant at 13 : 11 h, and water temperature was maintained at 26 ± 2◦C. We fed the fish ad libitum6 days per week with Hagen Nutrafin Basix flake cichlid food.
3.2. Study protocolAt the beginning of the study, we identified and captured the dominant breeding pair and thetwo subordinate helpers from each social group (n = 10). We sexed the fish by examination of theirexternal genitalia, measured (standard length and mass; table 1), and uniquely fin-clipped thembefore returning them to their home tanks. These fin clips are temporary, and the removed fin tissueregrows within a fortnight. Fin clipping does not adversely affect behaviour [51], and fish quicklyrecover from this procedure. We carried out two detailed behavioural observations per group onconsecutive days one week after measuring and marking the fish. All 10 groups were observed inrandom order on the same 2 days. Two trained observers observed each group once on each dayfor 10 min between 10.00 and 13.00. We recorded all social behaviours (aggressive, submissive andaffiliative) for all individuals in each group. The behaviours recorded are described in detail in anethogram for this species (table 2; adapted from [49,52]). Briefly, the behaviours we saw in our focalgroups were aggression, submission (submissive head-up posture, submissive displays, hook displays)and affiliation (follows, parallel swims, soft touches). We further subdivided the aggressive behaviourinto overt aggression (chases, rams, bites) and restrained aggression (head-down postures, frontaldisplays).
Immediately following the final behavioural observation, we quickly captured the focal fish, stunnedthem in an ice-water bath for 5–10 s and then killed them by spinal severance. We extracted the wholebrain from each fish within 2 min post-mortem. We weighed (table 1) and then immediately frozeeach brain on dry ice, and then stored them at −80◦C until the analyses of AVT and IT concentrationswere conducted. We confirmed the sex of each fish anatomically during dissection.
3.3. Brain isotocin and arginine vasotocin assaysWe determined the AVT and IT content in the brains of N. pulcher using HPLC-FL preceded by solid-phase extraction (SPE). The frozen brains were thawed and weighed before being sonicated in 1 mlMilli-Q water (MicrosonXL, Misonix, Farmingdale, NY). We added glacial acetic acid (3 µl) to thehomogenates, and then placed the samples in a boiling water bath for 3.5 min. The extracts werecooled on ice, and then centrifuged at 8000g for 20 min at 4◦C. We decanted the supernatants andloaded onto preconditioned (1 ml MeOH, 1 ml distilled water) SPE columns (30 mg ml−1, STRATA-X,Phenomenex, Torrance, CA). We passed water (600 µl) and then 0.1% trifluoroacetic acid (TFA) in 5%acetonitryl (600 µl) through the columns to wash away impurities, and then eluted the peptides by2 × 600 µl of 80% acetonitrile. The eluate was evaporated using Turbo Vap LV Evaporator (CaliperLife Science, Hopkinton, MA). We then froze the samples and stored them at −80◦C prior toHPLC analysis.
Before quantitative analysis, we re-dissolved the samples in 50 µl of 0.1% TFA in 30% acetonitrileand divided them into two aliquots. The derivatization of AVT and IT in each of the 25 µl samples waspreformed using 3 µl of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) solution (30 mg NBD-F in 1 mlof acetonitrile) in 25 µl phosphoric buffer (0.2 M, pH 9.0). We heated the solution at 60◦C for 3 min ina dry heating block and then cooled it down on ice before adding 5 µl of 1 M HCl. We measured thederivatized samples with Agilent 1200 Series Quaternary HPLC System (Agilent Technologies, SantaClara, CA). Chromatographic separation was achieved on a ZORBAX Eclipse XDB-C18 column (AgilentTechnologies, 150 × 4.6 mm I.D., 5 µm particle size). A gradient elution system was applied for separationof derivatized peptides. The mobile phase consisted of solvent A (0.1% TFA in H2O) and solvent B (0.1%TFA in acetonitrile : H2O [3 : 1]). A linear gradient was 40–65% of eluent B in 20 min. We set the flow rateat 1 ml min−1 and the column temperature at 20◦C. The injection volume was 58 µl. The fluorescencedetection was carried out at 530 nm with excitation at 470 nm.
Our procedure allowed us to determine the concentration of free AVT and IT after their dissociationfrom non-covalent complexes. This is important, because only this nonapeptide fraction binds tononapeptide receptors allowing them to act as neurotransmitters and/or neuromodulators in thebrain. This analytical procedure, which permits the measurement of biologically active nonapeptidesat their site of action, has been used successfully, with slight modifications, in several fish species[33–37,53].
................................................Table 2. The behaviours produced by Neolamprologus pulcher in the current study. (This ethogram is based on extensive laboratory andfield observations, and adapted from Sopinka et al. [52] and Hick et al. [49].)
frontal displays also called a puffed throat or an opercular flare.Focal fish extends out its opercula and lower jaw.Often associated with a posture where the head ispointed downwards
3.4. Statistical analysisWe summed all overt aggressive behaviours, restrained aggressive displays, submissive displays, andaffiliative behaviours produced by each individual across both observation periods and then calculatedthe rate of each class of behaviour produced per minute for each individual (table 1). We then ran linearmodels to explore the influence of each measured class of social behaviour produced per individual onthe levels of neuropeptides. We also examined the effect of sex (male or female) and dominance status(breeder or subordinate), on brain levels of AVT and IT. We initially included all two-way interactionsin the models, but none were significant (α = 0.05) and these were dropped from the final models. Socialgroup identity was included as a random effect in all models. Concentrations of AVT and IT werelog-transformed to meet the assumptions of the parametric tests. This study is correlational, and wecannot speak to the causal direction for the relationships we report. We treated our nonapeptide measuresas the response variable in the analyses and figures, because we collected these measurements after thebehavioural and demographic data. All analyses were performed using R v. 2.15.1.
4. ResultsBrain AVT concentrations were consistently higher in the subordinate than in the dominant N. pulcher(table 3 and figure 1a). There was a trend towards higher brain IT concentrations in the subordinatesrelative to dominant individuals and in males relative to females, but these trends did not reachsignificance (table 3 and figure 1b).
Affiliative behaviour was negatively related to brain IT levels (table 3 and figure 2h). There weremarginally non-significant trends towards negative relationships between submission and brain AVTlevels (table 3 and figure 2e), and restrained aggression and brain IT levels (table 3 and figure 2d).
................................................Table 3. Results of linear models exploring the influence of sex, status and behaviour on brain arginine vasotocin (AVT) and isotocin (IT)concentrations in Neolamprologus pulcher. (Two-way interaction terms were included in the original models, but none were significant(α = 0.05), and so these were dropped from the final models presented here. Values in italics indicate non-significant trends (0.1<p> 0.05). Values in bold-italics indicate model terms that significantly contribute to significant models (p< 0.05).)
There were no relationships between the rates of other behavioural categories, and brain AVT or ITconcentrations (table 3 and figure 2a–c,f,g).
5. DiscussionIn this study, we measured biologically available AVT and IT in the brains of dominant and subordinateN. pulcher of both sexes. In contrast to previous work measuring AVT gene expression in N. pulcher [47]and in zebrafish [26], subordinate N. pulcher had higher actual concentrations of bioavailable AVT intheir brains than did dominant fish. Measures of gene expression may not always correspond directlyto measures of final bioactive nonapeptide concentrations, in part, because there are several steps inbetween mRNA production and the resultant final products. For example, proisotocin and provasotocinare proteolytically cleaved into the final active hormones IT and AVT, respectively. Furthermore,differences in gene expression reflect differences in nonapeptide production, whereas the level ofavailable peptides may reflect differences in storage. For example, dominant and subordinate fish maydiffer in the degree to which AVT is being exported to the periphery versus being stored in the brain. InO. mossambicus, dominant individuals export a greater amount of AVT into their body where it stimulatesthe retention of urine, which is used as a social signal [34]. A similar effect could explain why we observedlower levels of AVT in the brains of dominant N. pulcher, despite the higher level of production suggestedby the greater AVT gene expression reported earlier [47]. Our finding that AVT levels were higher in
Figure 1. The relationship between (a) brain arginine vasotocin (AVT) and (b) brain isotocin (IT) concentration, and sex and dominancestatus in Neolamprologus pulcher. Subordinate N. pulcher have higher brain AVT concentrations. Boxes represent the interquartile range(i.e. the 25th–75thpercentile) of thedata,with themediandenotedby theband inside thebox.Whiskers represent the 10–90thpercentileof the data. Outliers are indicated. For sample sizes, see table 1. For full statistical details, see table 3.
subordinate animals appeared to be driven primarily by the male subordinates having particularly highAVT concentrations in their brains (figure 1a), although, in general, brain AVT was not sex-dependent,as the level of AVT did not differ between male and female dominants. The results of Aubin-Horth et al.[47], in which dominant N. pulcher had higher expression of the AVT gene, were driven primarily bydifferences in the female dominant fish, so this is another way in which our results differ from previousfindings using another technique. The fish used in this study came from the same study populationand were housed in the same laboratory under similar conditions as the fish used in Aubin-Horth et al.[47], suggesting that differences in the origin or handling of the animals do not underlie the apparentdifferences in the results of these two studies.
The higher levels of AVT in the whole brains of subordinate N. pulcher compared with dominantfish suggest that AVT could be involved in the expression of submissive behaviour in this species. Bothdominant and subordinate fish regularly produce aggressive behaviour, whereas submissive behaviouris rare in the dominants [46]. Therefore, the production of submissive displays is a key behaviouralfeature that distinguishes dominant from subordinate animals. AVT has been previously linked to theproduction of submissive behaviour in subordinates of another cichlid fish, the polygynous Africancichlid, A. burtoni, where greater AVT mRNA expression in the parvocellular region of the preoptic areawas found in subordinate males and the level of expression in this region correlated positively to the
Figure 2. The relationship between brain arginine vasotocin (AVT) and brain isotocin (IT) concentration and overtly aggressive (a,b),restrained aggressive (c,d), submissive (e,f ), and affiliative (g,h) behaviours inNeolamprologus pulcher. Affiliative behaviour is negativelyrelated to brain IT concentration as indicated by the regression line in panel (h). For sample sizes, see table 1. For full statistical details,see table 3.
................................................production of submissive behaviour [32]. Interestingly, we did not find a relationship between the level ofAVT in the brains on N. pulcher and the rate at which the fish produced submissive displays. Furthermore,there was a statistical trend towards a negative rather than towards a positive relationship between AVTlevels and submission. Collectively, these results suggest that a direct correspondence between AVT andsubmissive behaviour does not explain the status differences in AVT that we observed.
We did not find any association between the levels of biologically available IT and sex or social statusin N. pulcher. Furthermore, there was no relationship between the amount of IT in the brain and the rateat which submissive or aggressive behaviours were produced, although there was a trend towards anegative relationship between brain IT level and the production of restrained aggressive displays. Theseresults suggest that IT may not play a major role in the determination of social status in N. pulcher.Previous work on another cichlid, O. mossambicus, also did not find clear differences between the levelsof IT in brains of dominants and subordinates [34]. To the best of our knowledge, this is the firstmeasurement of brain IT in N. pulcher and adds to the small but growing literature on the behaviouralrole of IT in teleost fishes [18,19].
We found that the concentration of IT in the brain was correlated negatively with affiliative behaviourin N. pulcher. Affiliative behaviours involve approaching a social fellow, and interacting with thatconspecific within close spatial proximity. These results are consistent with the hypothesis that IT isan important regulator of social approach in fishes [20,50]. Reddon et al. [50] found that exogenousIT delivered intraperitoneally reduced the tendency for N. pulcher to approach and affiliate withconspecifics, whereas an oxytocin antagonist increased this tendency. The results of these administrationexperiments fit with our findings in this study. The IT appears to have a negative effect on socialapproach and affiliation in N. pulcher. By contrast, Thompson & Walton [20] found that IT increasedthe tendency for goldfish to approach a conspecific. The difference in results could suggest speciesdifferences in the role of this peptide in regulating social approach, or may result from differencesin methodology between the studies (intracerebroventricular infusions versus the measurement ofendogenous levels). The cause of this discrepancy in results will require future work to resolve, ideallyinvolving a combination of approaches on additional species. It is also worth considering that oxytocinhas a known anxiolytic effect in mammals [54–57] although it is unknown if the same effect occurs infishes. In many fish species, shoaling behaviour is an antipredator response [58], and shoaling may beevoked in stressful situations. If N. pulcher also cluster together with conspecifics in response to stress,then an anxiolytic effect of IT could, in principle, reduce the tendency to approach and affiliate withgroup members. This could partially account for our results and explain the reduced grouping tendencyfound in [50] following exogenous administration of IT. Further experimental study of the effects of ITon stress coping in N. pulcher in both social and non-social contexts will be essential to clarify this issue.Additional physiological measures, for instance, cortisol levels taken from the same animals would alsobe valuable.
We recorded all behaviour in terms of the number of discrete instances these behaviours wereproduced by each animal. Some of the behaviours (e.g. aggressive and submissive postures) that weobserved vary in their duration and therefore might be better described by the total duration of thebehaviour rather than by the frequency. In principle, gathering data on the duration of behavioursmay offer higher-resolution behavioural data for future studies looking at the relationship betweennonapeptide levels and behaviour in N. pulcher and other species. We also acknowledge that our studyis limited to 9–11 individuals per group and that this relatively small sample size may account for thelack of significant relationships that we detected between brain nonapeptide levels and many of thebehaviours we measured.
While the vast majority of studies of nonapeptide levels in the brains of fishes have usedneuroanatomical or genomic proxies of nonapeptide production [26,31,32,47,59], we directly measuredfree biologically available nonapeptides in the brains of our animals. The discrepancy in the patternsobserved between studies employing these different approaches emphasizes the complementary valueof looking directly at biologically available free nonapeptides along with genomic and neuroanatomicaltechniques. The discrepancy suggests that this method should be included in further study of the roleof nonapeptides in regulating behaviour [34]. One limitation of our current approach is that it involvesaveraging nonapeptide levels across the whole brain. The effects of the nonapeptides probably dependstrongly both on their site of origin and the activated target region [60]. It is known in fishes that AVTcells in the parvocellular and magnocellular regions of the preoptic area show different correlationswith behaviour [18,32]. Future studies that integrate multiple approaches within the same study systemalong with studies that provide brain-area-specific information about nonapeptide circuit function
................................................will be essential to fully elucidate the interplay between the production and circulating effects of thenonapeptide hormones and their role in regulating social behaviour.
In conclusion, dominant N. pulcher of both sexes have lower levels of biologically available AVTin their brains than do the subordinates. By contrast, we found no difference in the IT concentrationsacross dominance ranks. The individuals with higher concentrations of IT in their brains tended to beless affiliative than fish with lower levels of IT. Collectively, these results suggest that AVT may be animportant regulator of social status in this species, and support the notion that IT may be involved inregulating social approach. Our results emphasize the value of directly measuring biologically availablenonapeptide molecules in the brain.
Ethics statement. The procedures used in this study were approved by the Animal Research Ethics Board of McMasterUniversity (animal utilization protocol number 10-11-71) and adhered to the guidelines of the Canadian Council forAnimal Care, and the Animal Behaviour Society/Association for the Study of Animal Behaviour.Data accessibility. The dataset is available in the electronic supplementary material.Acknowledgements. We thank Will Swaney, Nadia Aubin-Horth, Michael Taborsky and an anonymous referee forproviding valuable feedback on earlier versions of this manuscript.Authors’ contributions. A.R.R., C.M.O., S.B. and E.K. conceived of the project and designed the study. A.R.R., C.M.O. andS.E.M.-R. collected behavioural data and brain samples. M.G. conducted the HPLC-FL analyses. C.M.O. analysed thedata. A.R.R. and C.M.O. wrote the first draft of the manuscript. All authors contributed to the revision of the finalmanuscript.Funding statement. This research was supported by a Natural Sciences and Engineering Research Council of CanadaDiscovery (NSERC) grant, Ontario Innovation Trust and Canadian Foundation for Innovation awards to S.B. aswell as National Science Centre (Poland) grant no. 2012/05/B/NZ4/02410 to E.K. and M.G. A.R.R. was supportedby the Margo Wilson and Martin Daly Ontario Graduate Scholarship and is currently supported by an NSERCPostdoctoral Fellowship and the Richard H. Tomlinson Postdoctoral Fellowship. C.M.O. was supported by the E.B.Eastburn Postdoctoral Fellowship from the Hamilton Community Foundation and is currently supported by anNSERC Postdoctoral Fellowship. S.B. is supported by the Canada Research Chair Programme.Competing interests. We have no competing interests.
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